This invention concerns the characterisation, classification, identification and typing of different DNA containing organisms such as plants, animals, bacteria and their viruses.
The science of detecting genomic polymorphisms is quickly evolving, and several techniques have been developed to compare the genomes of different organisms. These techniques utilise the whole genome or segments thereof for comparison purposes and are often referred to as DNA fingerprinting techniques. The main application fields for these techniques are gene mapping, detection of bacterial strain diversity, population analysis, epidemiology, gene expression and the demonstration of phylogenetic and taxonomic relationships.
In the application areas of bacterial identification and typing, pulsed-field gel electrophoresis, random amplified polymorphic DNA (RAPD) and DNA sequencing of different genes are frequently discussed as methods of the future, compared to traditional methods based on biochemical and growth properties. The drawback with all these and other suggested DNA fingerprinting methods is the use of electrophoresis. This is because electrophoresis is a laborious separation technology and the time from start to finish is long, ranging from 30 minutes up to more than 20 hours for pulsed-field gel electrophoresis, followed by both detection of the DNA and analysis of the results.
Since ribosomes are present in all living organisms, and the ribosomes contain three kinds of rRNA (in bacteria 5 S, 16 S, and 23 S), DNA sequencing of the corresponding genes is frequently used for characterisation, identification and taxonomy relations of bacteria, fungi and other organisms. The most widely used of the ribosomal genes is 16 S rDNA. The DNA sequences of these genes contain well-defined segments of different evolutionary variability regions, which in the 16 S rRNA molecule are referred to as universal, semi-conserved and variable regions. Oligbnucleotide primers complementary to universal regions can be used for amplification of ribosomal RNA from any organism or bacteria, the generated ribosomal fragment being then sequenced. In a computer search against a database with all known ribosomal sequences the species can be assigned.
Random amplified polymorphic DNA (RAPD) is a process for detecting polymorphisms on the basis of nucleotide differences and is covered by U.S. Pat. No. 5,126,239 of Livak et al, 1992. RAPD analysis is one of the most sensitive, reproducible and efficient methods currently available in the research field for distinguishing different strains and isolates of a species. RAPD analysis is a technique that uses a single short oligonucleotide primer of arbitrary sequence in a low stringency amplification reaction (Welsh J and McClelland M, 1990, Nucleic Acids Research, 18 (24), 7213-7218; Williams J G et al (1990) Nucleic Acids Research, 18, 6531-6535). The generated DNA fragments are subsequently analysed by gel electrophoresis. Analysis can either be done automatically on line with an automated DNA sequencer or on any electrophoresis gel and stained with ethidium bromide or silver.
In arrayed primer extension techniques (APEX), primers which have hybridised with a template, having a free 3'-end and having a free hydroxyl group, can be extended with free dNTPs and a DNA polymerase. If ddNTPs or other chain terminators are added to the mixture the elongation will terminate at that point. A similar method to this was developed for mutation detection (WO 91/13075). The authors used a PCR template bound to wells of a microtitre plate, and the primers were added for extension after binding to the template.
Further development of this method was carried out where consecutive primers overlapping each other by one base were bound to a support in the form of an array (also called a DNA chip). These were bound to the surface by the 5'-end, so that the 3'-end was free for elongation after addition of template, ddNTP and a polymerase. The chain terminating molecules were labelled, with any type of chromophore, fluorophore, isotope or antibody reactive reagent. This technique is described in WO 95/00669.